Compositions and methods for treating celiac sprue disease

ABSTRACT

Polypeptides, and methods for their use, are disclosed that have an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:1, are provided, wherein (a) the polypeptide degrades a PFQPQLPY (SEQ ID NO: 140) peptide and/or a PFPQPQQPF (SEQ ID NO: 68) at pH 4; (b) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and (c) the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424, 449, 461, 463, 105, 171, 172, 173, 174, and 456.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.15/575159 filed Nov. 17, 2017, which is a national stage filing of PCTApplication Serial No. PCT/US2016/036356 filed Jun. 8, 2016, whichclaims priority to U.S. Provisional Patent Application Ser. No.62/172,557 filed Jun. 8, 2015, each incorporated by reference herein inits entirety.

BACKGROUND

Celiac sprue is a highly prevalent disease in which dietary proteinsfound in wheat, barley, and rye products known as ‘glutens’ evoke animmune response in the small intestine of genetically predisposedindividuals. The resulting inflammation can lead to the degradation ofthe villi of the small intestine, impeding the absorption of nutrients.Symptoms can appear in early childhood or later in life, and rangewidely in severity, from diarrhea, fatigue and weight loss to abdominaldistension, anemia, and neurological symptoms. There are currently noeffective therapies for this lifelong disease except the totalelimination of glutens from the diet. Although celiac sprue remainslargely underdiagnosed, its' prevalence in the US and Europe isestimated at 0.5-1.0% of the population. In addition to celiac sprue, asignificant fraction of the population is thought to suffer from thecondition of non-celiac gluten sensitivity (NCGS), which is caused bythe ingestion of gluten but is mechanistically distinct from celiacdisease, though the symptoms are frequently indistinguishable from thoseof celiac sprue. The identification of suitable naturally-occurringenzymes as oral therapeutics for celiac disease and NCGS is difficultdue to the stringent physical and chemical requirements to specificallyand efficiently degrade gluten-derived peptides in the harsh and highlyacidic environment of the human digestive tract. Since gluten peptidesinitiate the immune response immediately upon entering the intestines,it is imperative that any oral enzyme therapeutic for celiac diseasebreak down these immunogenic gluten regions in the gastric compartment,thereby preventing these gluten peptides from causing intestinal damagedue to inflammation.

SUMMARY OF THE INVENTION

In one aspect, the invention provides polypeptides comprising an aminoacid sequence at least 75%, 80%, 85%, 90%, 95%, or more identical to theamino acid sequence of SEQ ID NO: 1, wherein

(a) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 1 atone or more residues selected from the group consisting of 463, 221,262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402,406, 424, 449, 461, 105, 171, 172, 173, 174, and 456.

In one embodiment, the polypeptide comprises an amino acid change fromSEQ ID NO: 1 at one or more residues selected from the group consistingof 463, 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T,368F/Q, 399, 402, 406, 424, 449, and 461. In another embodiment, thepolypeptide comprises an amino acid change from SEQ ID NO: 1 at one ormore residues selected from the group consisting of 221D/N/Q/H, 262E,268S/T/A, 269L/T, 270A/T/V, 319A, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399Q,402S/Q, 406S, 424K, 449E/N/Q, 461R, and 463A/L/M/Q/R/T/V. In a furtherembodiment, the polypeptide comprises an amino acid change from SEQ IDNO: 1 at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more residuesselected from the group.

In one embodiment, the polypeptide comprises amino acid changes from SEQID NO: 1 at residues 399 and 449, such as amino acid changes 399Q and449Q. In another embodiment, the polypeptide comprises amino acidchanges 358S and 463T. In a further embodiment, the polypeptidecomprises amino acid changes 262E, 269T, 354Q, 358S, 399Q, 449Q, and463T. In another embodiment, the polypeptide comprises amino acidchanges 319A, 368F, 399Q, 449Q, and 463T. In a further embodiment, thepolypeptide comprises amino acid changes 262E, 269T, 270V, 354Q, 358S,399Q, and 449Q. In a still further embodiment, the polypeptide comprisesamino acid changes 262E, 269T, 320M, 354Q, 358S, 399Q, 449Q, and 463T.In another embodiment, the polypeptide comprises amino acid changes319A, 320M, 368F, 399Q, 449Q, and 463T. In one embodiment, thepolypeptides comprise an amino acid change from SEQ ID NO: 1 at one ormore amino acid positions selected from the group consisting of 105,171, 172, 173, 174, and 456, such as amino acid changes 105H; 171R A, orS; 172R, A, or S; 173R or S, 174S, and/or 456V.

In another aspect, the invention provides polypeptide comprising anamino acid sequence at least 75%, 80%, 85%, 90%, 95%, or more identicalto the amino acid sequence of SEQ ID NO: 71, wherein

(a) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 71 atone or more residues selected from the group consisting of 274, 32, 73E,79, 80, 81, 130A, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210, 213, 217, 235, 260,267, and 272.

In one embodiment, the polypeptide comprises an amino acid change fromSEQ ID NO: 71 at one or more residues selected from the group consistingof 274, 32, 73E, 79, 80, 81, 130A, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210,213, 217, 235, 260, and 272.

In one embodiment, the polypeptide comprises an amino acid change fromSEQ ID NO: 71 at one or more residues selected from the group consistingof 32D/N/Q/H, 73E, 79S/T/A, 80L/T, 81A/T/V, 130A, 165E/Q/R/Y, 169S/Q/T,179F/Q, 210Q, 2135/Q, 217S, 235K, 260E/N/Q, 272R, and 274A/L/M/Q/R/TN.In a further embodiment, the polypeptide comprises an amino acid changefrom SEQ ID NO: 71 at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or moreresidues selected from the group. In one embodiment, the polypeptidecomprises amino acid changes from SEQ ID NO: 71 at residues 210 and 260,including but not limited to amino acid changes 210Q and 260Q. Inanother embodiment, the polypeptide comprises amino acid changes 169Sand 274T. In a further embodiment, the polypeptide comprises amino acidchanges 73E, 80T, 165Q, 169S, 210Q, 260Q, and 274T. In anotherembodiment, the polypeptide comprises amino acid changes 130A, 179F,210Q, 260Q, and 274T. In a further embodiment, the polypeptide comprisesamino acid changes 73E, 80T, 81V, 165Q, 169S, 210Q, and 260Q. In a stillfurther embodiment, the polypeptide comprises amino acid changes 73E,80T, 320M, 165Q, 169S, 210Q, 260Q, and 274T. In one embodiment, thepolypeptide comprises amino acid changes 130A, 131M, 179F, 210Q, 260Q,and 274T. In another embodiment, the polypeptide comprises an amino acidchange from SEQ ID NO: 71 at amino acid position 267, including but notlimited to 267V.

In another embodiment, the polypeptide comprises the amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-42, 44-60 and72-112, and 114-130 and 150-155. In a further embodiment, thepolypeptide comprises the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-42, 55-60 and 72-112, and 125-130 and150-155.

In one embodiment, the polypeptides of the invention may furthercomprise a histidine tag at the C-terminus of the polypeptide. Inanother embodiment, the histidine tag comprises a cleavable histidinetag In a specific embodiment, the cleavable histidine tag may compriseor consist of the amino acid sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO:139). In another embodiment, the cleavable histidine tag may comprisethe amino acid sequence X_(N)PQ(L/Q)PX_(N)HHHHHH (SEQ ID NO: 131),wherein X_(N) is an linker of between 1-25 amino acid residue. In afurther embodiment, the cleavable histidine tag may comprise the aminoacid sequence GSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 132).

In another aspect, the invention provides nucleic acids encoding thepolypeptide of any embodiment of the invention. The invention furtherprovides nucleic acid expression vector comprising the nucleic acids ofthe invention. The invention further provides recombinant host cellscomprising the nucleic acid expression vectors of the invention. Theinvention also provides pharmaceutical composition, comprising thepolypeptide, nucleic acid, nucleic acid expression vector, and/or therecombinant host cell of any embodiment of the invention, and apharmaceutically acceptable carrier.

In another aspect, the invention provides methods for treating celiacsprue or non-celiac gluten sensitivity (NCGS), comprising administeringto an individual with celiac sprue or NCGS an amount effective to treatthe celiac sprue or NCGS of a polypeptide or pharmaceutical compositionof any embodiment of the invention. In one embodiment, the polypeptideor the pharmaceutical composition is administered orally.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Kuma030 is capable of rapidly and effectively degrading theimmunogenic regions of gluten in gastric conditions. (A) The amount ofgluten detected after a 60 minute incubation period with the indicatedconcentrations of either EPB2 and SCPEP (at a 1:1 ratio), Kuma010, orKuma030 in gastric conditions, as measured by ELISA using the G12antibody. The starting concentration of gluten was 10 mg mL-1 (10,000ppm). Note that the Y-axis is plotted on a logarithmic scale. (B) Theamount of gluten detected at 5 or 30 minutes after incubation with EPB2and SCPEP at 400 μg mL-1, or Kuma030 at 10, 100, or 400 μg mL-1. Thestarting concentration of gluten was 10 mg mL-1. Samples were normalizedto the amount of gluten measured after incubation with pepsin alone. ES:a 1:1 weight ratio of EPB2 and SCPEP. Asterisk indicates that the amountof gluten was below the level of quantification (5 ppm). (C) HPLC traceof full-length peptide (grey dotted lines) or breakdown products (blacklines) of the immunodominant peptides from gliadin (W02-E07, W03-E07,33mer), hordein (B08-E2E7), or secalin (R11-E4E7). The bottom of thefigure shows the amino acid sequence of the 33mer peptide, position ofknown immunogenic epitopes (horizontal lines), location of the Kuma030cleavage sites as determined by LCMS (vertical lines), and elution peaksof the resulting breakdown products (grey arrows). Immunodominantpeptides displayed the following breakdown patterns: B08-E2E7:PQQPIPQ∥QPQPYPQ∥Q (SEQ ID NO: 61); R11-E4E5: QPFPQ∥QPEQIIPQ∥QP (SEQ IDNO:62); W02-E7: LQPFPQPQ∥LPYPQPQ (SEQ ID NO: 63); W03-E7:QPFPOPQ∥QPFPWQP (SEQ ID NO: 64). All peptide masses and elution timeswere confirmed by LCMS. Note that although the undigested W03-E07peptide eluted at approximately the same time as the W03-E07 breakdownfragments, these are separate peaks, as determined by LCMS. aAU,arbitrary absorbance units.

FIG. 2. Gliadin treated with Kuma030 loses it immunostimulatorypotential. (A-E) Purified gliadin was treated with Kuma030 at thespecified concentration for 60 minutes at pH 4.0 at 37° C. in thepresence of 0.6 mg mL-1 pepsin. After the gastric phase, the pH of thesamples was increased, and samples were treated with chymotrypsin andTG2. Samples were then exposed to T cell lines from patients #1 (A), #2(B), #3 (C), #4 (D), or #5 (E), in the presence of autologous irradiatedB cell lines, and IFN-γ was measured by ELISA. Phytohemagglutenin (PHA)and a peptic-tryptic digest of gliadin (PT-Gliadin) were included aspositive controls. Incubation of T cell lines with antigen-presentingcells in the absence of antigens acted as a negative control. (F) T cellstimulatory epitopes recognized by the T cells used in this assay andthe predicted Kuma030 cleavage sites within these epitopes. PredictedKuma030 cleavage sites are shown by a vertical Cleavage sites arepredicted based on Kuma030 activity on gliadin peptides as presented inFIG. 1F (from top to bottom: SEQ ID NOs: 141, 142, 141, 142, 143, 144,141, 142, 143, 144, 145, 145, 146, 145).

FIG. 3. Kuma030 is not toxic to T cells. Possible toxic effect ofenzyme-treated gliadin was assessed in human peripheral bloodmononuclear cells (PBMCs) from healthy donors that were stimulated withthe mitogen phytohemagglutinin (PHA). (a,b) IFN-g production of T cellsfrom healthy donor #1 (A) or healthy donor #2 (B). (C,D) T cellproliferation of T cells from healthy donor #1 (C) or healthy donor #2(D). No effect on IFN-g production or cell proliferation was observed,except in the proliferation of healthy donor #2 (but not in theproduction of IFN-g). Since the reduction of PHA-stimulated cellproliferation was the same irrespective of the enzyme treatment, weconclude that this effect was not due to toxicity of the samples.

FIG. 4. Kuma030 efficiently degrades gluten in complex food matrices.(A) The amount of gluten remaining in whole wheat bread after a 30minute incubation with either Kuma030 or a 1:1 combination of EPB2:SCPEPat the indicated enzyme concentrations. The starting concentration ofgluten was 10,000 ppm. Note both axes are plotted on a logarithmicscale. (B) The amount of gluten remaining in a wheat beer afterincubation with the indicated concentrations of either a 1:1 ratio ofEPB2:SCPEP, or Kuma030, at 37° C. or 4° C., at 5, 15, or 60 minutes.Gluten was detected using the G12 ELISA method.

FIG. 5. Comparison of 26-mer and 33-mer degradative abilities ofKuma010, Kuma020, Kuma030, Kuma040, and Kuma050. Enzymes were incubatedat 2 μg mL⁻¹ with 1 mg mL⁻¹ of either the 33mer peptide (FIG. 5a ) orthe 26mer peptide (FIG. 5b ) for 60 minutes. Samples were taken at theindicated timepoints, and the concentrations of peptide breakdownproducts were measured. Breakdown products were: from the 33mer peptide(SEQ ID NO: 69), LPYPQPQF (SEQ ID NO: 137); from the 26mer peptide (SEQID NO: 70), QPYPQ (SEQ ID NO: 147). Activities are graphed as the ratioof the appropriate m/z signal to that of the internal standard.

FIG. 6. The G320M mutation improves activity by 2-4 fold. Shown isactivity in degradation of immunogenic epitopes DQ2.5-glia-α1a (containsPQL) (FIG. 6a ), and DQ2.5-glia-ω1 (contains PQQ) (FIG. 6b ). Sampleswere taken at the indicated timepoints, and the concentration of peptidebreakdown product PFPQPQ (SEQ ID NO: 148) was measured. Activities aregraphed as the ratio of the appropriate m/z signal to that of theinternal standard.

FIG. 7. P171R and H172R mutations do not appear to negatively affectactivity. Enzymatic activities are shown for enzyme variants Kuma030,Kuma031, Kuma032, Kuma040, Kuma041, and Kuma042 on the immunogenicgliadin epitope DQ2.5-glia-α1a. Samples were taken at the indicatedtimepoints, and the concentration of peptide breakdown product PFPQPQ(SEQ ID NO: 148) was measured. Activities are graphed as the ratio ofthe appropriate m/z signal to that of the internal standard. All enzymeswere purified by anion exchange.

FIG. 8. Kuma062 demonstrates a higher level of enzymatic activity thanKuma030 or Kuma040. Shown is activity on the immunogenic gliadin epitopeDQ2.5-glia-ω1. FIG. 8a : detection of full-length DQ2.5-glia-ω1; FIG. 8b: detection of the PFPQPQ (SEQ ID NO: 148) breakdown product. Sampleswere taken at the indicated timepoints, and the concentration of eitherfull-length peptide, or breakdown product PFPQPQ (SEQ ID NO: 148), wasmeasured. Activities are graphed as the ratio of the appropriate m/zsignal to that of the internal standard.

DETAILED DESCRIPTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, amino acid residues are abbreviated as follows: alanine(Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg;R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides polypeptidescomprising an amino acid sequence at least 75% identical to the aminoacid sequence of SEQ ID NO:1, wherein

(a) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 1 atone or more residues selected from the group consisting of 221, 262E,268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406,424, 449, 461, 463, 105, 171, 172, 173, 174, and 456. In one embodiment,the polypeptide comprises an amino acid change from SEQ ID NO: 1 at oneor more residues selected from the group consisting of 221, 262E, 268,269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424,449, 461, and 463.

SEQ ID NO: 1 (Kuma 011) Kuma011 (SEQ ID NO: 1)MSDMEKPWKE(10)GEEARAVLQG(20)HARAQAPQAV(30)DKGPVAGDER(40)MAVTVVLRRQ(50)RAGELAAHVE(60)RQAAIAPHAR(70)EHLKREAFAA(80)SHGASLDDFA(90)ELRRFADAHG(100)LALDRANVAA(110)GTAVLSGPDD(120)AINRAFGVEL(130)RHFDHPDGSY(140)RSYLGEVTVP(150)ASIAPMIEAV(160)LGLDTRPVAR(170)PH(172)FRMQRRAE(180)GGFEARSQ(188)AA(190)APTAYTPLDV(200)AQAYQFPEGL(210)DGQGQCIAII(220)E(221/32)LGGGYDEAS(230/41)LAQYFASLGV(240/51)PAPQVVSVSV(250/61)DGASNQPTGD(260/71)PK(262/73)GPDGE(267/78)V(268/79)E(269/80)L(270/81)D(271/82)IEVAGALAP(280/91)GAKFAVYFAP(290/101)DTTAGFLDAI(300/111)TTAIHDPTLK(310/121)PSVVSISWS(319/130)G(320/131)PEDSWTSAAI(330/141)AAMNRAFLDA(340/151)AALGVTVLAA(350/161)AGDS(354/165)GSTG(358/169)GE(360/171)QDGLYHVH(368/179)FP(370/181)AASPYVLACG(380/191)GTRLVASGGR(390/201)IAQETVWND(399/210)G(400/211)PD(402/213)GGAT(406/217)GGGV(410/221)SRIFPLPAWQ(420/231)EHAN(424/235)VPPSAN(430/241)PGASSGRGVP(440/251)DLAGNADPA(449/260)T(450/261)GYEVVIDGEA(460/271)T(461/272)VI(463/274)GGTS(467/278)AVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQA LLPSASQPQP

Kuma010 as reference herein is identical to Kuma011, but includes thehistidine tag sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139) at itsC-terminus.

Bold face residues represent the N-terminal portion present in theunprocessed polypeptide; non-bold faced font represents residues presentin the processed version of the polypeptide. The numbers in parenthesesindicate residue number; where there are two numbers separated by a “/”,the number on the left is the residue number in the unprocessed version,and the number on the right is the residue number in the processedversion. SEQ ID NO:1 is the unprocessed version of Kuma011; SEQ ID NO:71is the processed version of Kuma011.

As disclosed in the examples that follow, polypeptides according to thisaspect of the invention are improved polypeptides for use, for example,in treating celiac sprue. The polypeptides are modified versions ofeither the processed version or the preprocessed version of thepolypeptide of SEQ ID NO: 1 (KUMAMAX™, hereinafter referred to asKuma010), which was disclosed as useful for treating celiac sprue(WO2013/023151). Polypeptides for treating celiac sprue are capable ofdegrading proline (P)- and glutamine (Q)-rich components of gluten knownas ‘gliadins’ believed responsible for the bulk of the immune responsein most celiac sprue patients. The polypeptides of the present inventionshow superior activity in degrading peptides having a PQLP (SEQ ID NO:65) or PQQP (SEQ ID NO: 66) motif (such as PFPQPQLPY (SEQ ID NO: 67)and/or PFPQPQQPF (SEQ ID NO: 68)), which are substrates representativeof gliadin) at pH 4 compared to Kuma011 and other polypeptides disclosedas useful for treating celiac sprue (WO2015/023728), and/or are shown toimprove production of the polypeptides. Thus, the polypeptides of theinvention constitute significantly improved therapeutics for treatingceliac sprue.

Thus, the polypeptides of the invention degrade a PFPQPQLPY (SEQ ID NO:67) peptide and/or a PFPQPQQPF (SEQ ID NO: 68) peptide at pH 4, as wellas LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69) and/orFLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70).

Polypeptides of the first aspect of the invention comprise preprocessedversions of the polypeptide enzymes of the invention.

In a second aspect, the invention provides polypeptides comprising anamino acid sequence at least 75% identical to the amino acid sequence ofSEQ ID NO:71, wherein

(a) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 71 atone or more residues selected from the group consisting of 32, 73E, 79,80, 81, 130A, 131, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210, 213, 217, 235,260, 267, 272, and 274. In one embodiment, the polypeptide comprises anamino acid change from SEQ ID NO: 71 at one or more residues selectedfrom the group consisting of 32, 73E, 79, 80, 81, 130A, 131, 165E/Q/R/Y,169S/Q/T, 179F/Q, 210, 213, 217, 235, 260, 272, and 274.

Polypeptides of the first aspect of the invention comprise processedversions of the polypeptide enzymes of the invention, and also degrade aPFPQPQLPY (SEQ ID NO: 67) peptide and/or a PFPQPQQPF (SEQ ID NO: 68)peptide at pH 4, as well as LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ IDNO: 69) and/or

FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70).

As used herein, “at least 75% identical” means that the polypeptidediffers in its full length amino acid sequence by 25% or less (includingany amino acid substitutions, deletions, additions, or insertions) fromthe polypeptide defined by SEQ ID NO: 1 or SEQ ID NO: 71.

In various embodiments of any aspect of the polypeptides of theinvention, the polypeptides comprise or consist of an amino acidsequence at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence according to SEQ ID NO: 1(preprocessed) or SEQ ID NO:71 (processed).

The polypeptide of any aspect of the polypeptides of the invention maycomprises an amino acid change from SEQ ID NO: 1 or SEQ ID NO:71 at 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or all 24 (depending on the embodiment) of the recited residues.

In one embodiment of the polypeptides of the first aspect of theinvention, the polypeptide comprises one or more amino acid changes fromSEQ ID NO: 1 at one or more residues selected from the group consistingof 221D/N/Q/H, 262E, 2685/T/A, 269L/T, 270A/T/V, 319A, 354E/Q/R/Y,358S/Q/T, 368F/Q, 399Q, 402S/Q, 406S, 424K, 449E/N/Q, 461R, and463A/L/M/Q/R/T/V. As used throughout, the number indicates the residuenumber in the SEQ ID NO:1 or SEQ ID NO:71 polypeptide sequence, and thesingle letter amino acid abbreviations to the right of the numberindicate the possible amino acid substitutions compared to the aminoacid residue present at that position in SEQ ID NO:1 or 71.

In another embodiment of the polypeptides of the first aspect of theinvention, the polypeptide comprises amino acid changes from SEQ ID NO:1 at residues 399 and 449. In one embodiment, the polypeptide comprisesamino acid changes 399Q and 449Q.

In a further embodiment of the polypeptides of the first aspect of theinvention, the polypeptide comprises 358S and 463T. These polypeptideare extensively characterized in the examples that follow, asexemplified by the polypeptide designated as Kuma020 and variantsthereof.

In one embodiment of the polypeptides of the first aspect of theinvention, the polypeptide comprises 262E, 269T, 354Q, 358S, 399Q, 449Q,and 463T. These polypeptide are extensively characterized in theexamples that follow, as exemplified by the polypeptide designated asKuma030 and variants thereof. In another embodiment of the polypeptidesof the first aspect of the invention, the polypeptide comprises 319A,368F, 399Q, 449Q, and I463T. These polypeptide are extensivelycharacterized in the examples that follow, as exemplified by thepolypeptide designated as Kuma040 and variants thereof. In a furtherembodiment of the polypeptides of the first aspect of the invention, thepolypeptide comprises 262E, 269T, 270V, 354Q, 358S, 399Q, and A449Q.These polypeptide are extensively characterized in the examples thatfollow, as exemplified by the polypeptide designated as Kuma050 andvariants thereof. In one embodiment of the polypeptides of the firstaspect of the invention, the polypeptide comprises 262E, 269T, 320M,354Q, 358S, 399Q, 449Q, and 463T. These polypeptide are extensivelycharacterized in the examples that follow, as exemplified by thepolypeptide designated as Kuma060 and variants thereof. In a stillfurther embodiment of the polypeptides of the first aspect of theinvention, the polypeptide comprises, 319A, 320M, 368F, 399Q, 449Q, and463T. These polypeptide are extensively characterized in the examplesthat follow, as exemplified by the polypeptide designated as Kuma070 andvariants thereof.

In another embodiment of the polypeptides of the first aspect of theinvention, the polypeptides comprise an amino acid change from SEQ IDNO: 1 at one or more amino acid positions selected from the groupconsisting of 105, 171, 172, 173, 174, and 456. In one embodiment, theamino acid change is 105H; 171R A, or S; 172R, A, or S; 173R or S, 174S,and/or 456V. In another embodiment, the amino acid change is 171R, 172R,and/or 456V.

In one embodiment of the polypeptides of the second aspect of theinvention the polypeptide comprises one or more amino acid change fromSEQ ID NO: 71 at one or more residues selected from the group consistingof 32D/N/Q/H, 73E, 795/T/A, 80L/T, 81A/TN, 130A, 165E/Q/R/Y, 169S/Q/T,179F/Q, 210Q, 213S/Q, 217S, 235K, 260E/N/Q, 272R, and 274A/L/M/Q/R/T/V.In another embodiment of the polypeptides of the second aspect of theinvention, the polypeptide comprises amino acid changes from SEQ ID NO:71 at residues 210 and 260. In a further embodiment of the polypeptidesof the second aspect of the invention, the polypeptide comprises aminoacid changes 210Q and 260Q. In one embodiment of the polypeptides of thesecond aspect of the invention, the polypeptide comprises 169S and 274T.(Kuma 20 genus) In another embodiment of the polypeptides of the secondaspect of the invention the polypeptide comprises 73E, 80T, 165Q, 169S,210Q, 260Q, and 274T. (Kuma 30 genus) In a further embodiment of thepolypeptides of the second aspect of the invention, the polypeptidecomprises 130A, 179F, 210Q, 260Q, and 274T. (Kuma 40 genus) In a stillfurther embodiment of the polypeptides of the second aspect of theinvention, the polypeptide comprises 73E, 80T, 81V, 165Q, 169S, 210Q,and 260Q. (Kuma 50 genus) In one embodiment of the polypeptides of thesecond aspect of the invention, the polypeptide comprises 73E, 80T,320M, 165Q, 169S, 210Q, 260Q, and 274T. (Kuma 60 genus) In anotherembodiment of the polypeptides of the second aspect of the invention,the polypeptide comprises 130A, 131M, 179F, 210Q, 260Q, and 274T. (Kuma70 genus) In a still further embodiment of the polypeptides of thesecond aspect of the invention, the polypeptides comprise an amino acidchange from SEQ ID NO: 71 at one or more amino acid positions selectedfrom the group consisting of 267. In one embodiment, the amino acidchange is, 267V.

In a further embodiment of the polypeptides of any aspect of theinvention, the polypeptides further comprise a histidine tag at theC-terminus of the polypeptide, to facilitate isolation of thepolypeptide. Any suitable histidine tag can be used; in one embodimentthe tag is linked to a TEV protease cut sit (ENLYFQS) (SEQ ID NO: 149)to allow for its efficient removal with TEV protease after purification,for example, the tag may comprise or consist of the amino acid sequenceGSTENLYFQSGALEHHHHHH (SEQ ID NO: 139). In another embodiment, thehistidine tag is a. cleavable histidine tag, permitting easier removalof the His-tag. In one embodiment, the cleavable histidine tag comprisesthe amino acid sequence X_(N)PQ(L/Q)PX_(N)HHHHHH (SEQ ID NO: 131),wherein X_(N) is an linker of between 1-25 amino acid residues. In onenon-limiting example, the cleavable histidine tag comprises the aminoacid sequence GSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 132).

In one embodiment of any aspect of the polypeptides of the invention,amino acid substitutions compared to SEQ ID NO: 1 or SEQ ID NO: 71 maycomprise one or more of the substitutions noted in Tables 1 or 2.Substitutions at these positions were found to be generallywell-tolerated (i.e. generally result in minor to no effects onactivity), and in some cases to increase the activity of thepolypeptides of the invention by no more than 20%.

TABLE 1 Possible Amino Acid Substitutions at Position Relative toKuma10. Residue number (preprocessed/processed) Residue 221/32 D, N, Q,H 261/72 A, R, N, D, C, Q, E, G, H, I, L, K, M, S, T, W, Y, V 262/73 A,R, N, D, C, Q, E, G, H, I, L, M, F, T, W, Y, V 264/75 A, N, D, C, Q, E,G, S, T, Y 266/77 A, C, S 268/79 S, T 269/80 L, T 270/81 A, R, N, D, C,Q, E, G, I, K, S, T, V 317/128 A, N, C, G, T, V 318/129 A, R, N, D, C,Q, E, G, H, L, K, M, F, S, T, Y, V 319/130 A, N, D, C, Q, H, M, T320/131 A, R, N, D, C, Q, K, M, S 350/161 N, D, C, G, S, T 351/162 G, S353/164 A, R, N, C, Q, E, G, I, K, M, S, T, V 354/165 A, R, N, D, C, Q,E, G, H, L, K, M, F, T, W, Y 358/169 A, S, N, Q, T 368/179 A, R, N, C,Q, E, G, K, M, F, S, T, W, Y 397/208 A, C, F, Y 399/210 Q, N 402/213 Q,N, S 406/217 S 424/235 K 446/257 G, S 448/259 A, R, N, D, C, Q, E, G, H,I, L, K, M, F, S, T, W, Y, V 449/260 Q, E, G, N 456/267 A, N, D, C, Q,E, G, H, L, S, T, V 461/272 R 463/274 A, R, N, D, C, Q, E, G, H, L, K,M, F, S, T, W, Y, V 464/277 A, N, D, C, S, 466/279 D, C, G, S

In another embodiment of any aspect of the polypeptides of theinvention, amino acid substitutions compared to SEQ ID NO: 1 or SEQ IDNO: 71 may comprise one or more of the substitutions noted in Table 2.

Residue number (preprocessed/processed) Residue 221/32 D, N, Q, H 261/72S 262/73 A, R, N, D, Q, E, G, L, M, T 264/75 A 268/79 S, T 269/80 L, T270/81 A, T, V 317/128 A, T 319/130 A 354/165 A, R, N, D, Q, E, K, T, Y358/169 A, S, N, Q, T 368/179 A, N, D, Q, E, S, T 402/213 Q, S 406/217 S424/235 K 446/257 S 449/260 Q, N, A 456/267 V 461/272 R 463/274 A, R, Q,L, M, T, V

In another embodiment of any aspect of the polypeptides of theinvention, amino acid at each residue of the polypeptides of theinvention may be as noted in Table 3, which lists all of the possiblemutations at each position in the polypeptide enzymes as predicted bycomputational mutagenesis analysis. As described in the examples thatfollow, mutations were tested at each position found in the active site(residues 261-264, 266-267, 270, 317-320, 350-354, 368, 397, 403-404,446, 448, 456, and 463-468) using degenerate primers to test the effectsof various amino acid substitutions on activity; those that did notinterfere with activity can be incorporated in the polypeptides of theinvention, as reflected in Table 3.

TABLE 3 Possible Amino Acids at Residues Relative to Kuma 010 FullLength Mature Amino Acid Possibilities 190 1 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR,VAL 191 2 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET,PHE, PRO, SER, THR, TRP, VAL 192 3 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, LEU, LYS, MET, PHE, PRO, SER, TRP, TYR 193 4 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR,TRP, TYR, VAL 194 5 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU,LYS, MET, PHE, SER, THR, TRP, TYR, VAL 195 6 ALA, ASN, CYS, GLN, HIS,LEU, MET, PHE, THR, TYR 196 7 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, LYS, MET, PHE, SER, THR, TRP, TYR 197 8 ALA, GLY, PRO, SER 198 9ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL 199 10 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 200 11 ALA, ASN, ASP, CYS,GLY, ILE, SER, THR, VAL 201 12 ALA, CYS, GLY, SER 202 13 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP,TYR, VAL 203 14 ALA, GLY, SER 204 15 ALA, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR 205 16 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR,VAL 206 17 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, SER, THR, TYR, VAL 207 18 ALA, CYS, GLN, GLU, GLY, LYS, PRO, SER,THR, TRP 208 19 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU,LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 209 20 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR,VAL 210 21 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER, THR,VAL 211 22 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE,SER, THR, TYR 212 23 GLY 213 24 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 214 25 GLY 215 26 ALA,ASN, ASP, CYS, GLN, GLU, GLY, SER, THR 216 27 ALA, ASN, ASP, CYS, GLN,GLY, SER, THR, VAL 217 28 ALA, CYS, ILE, LEU, SER, THR, VAL 218 29 ALA,GLY, SER 219 30 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, MET,SER, THR, VAL 220 31 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR,VAL 221 32 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR, VAL 222 33ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, SER, THR, VAL 223 34ALA, ARG, ASN, ASP, CYS, GLU, GLY, LYS, MET, SER 224 35 GLY 225 36 GLY226 37 ALA, ARG, ASN, ASP, CYS, GLU, GLY, HIS, LEU, PHE, SER, THR, TRP,TYR 227 38 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LYS, MET, SER 228 39ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,PRO, SER, THR, TRP, TYR, VAL 229 40 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 230 41 ALA,GLY, SER 231 42 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, SER, THR 232 43ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL, 233 44 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 234 45 ALA, ASN,CYS, GLY, HIS, PHE, SER, TYR 235 46 ALA, ASN, ASP, CYS, HIS, MET, PHE,SER, THR, TRP, TYR 236 47 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS,ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 237 48 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP,TYR, VAL, 238 49 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU,MET, SER, THR, VAL 239 50 GLY 240 51 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, LEU, LYS, MET, SER, THR, TYR, VAL 241 52 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP,TYR, VAL 242 53 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS,MET, PRO, SER, THR, VAL 243 54 ALA, GLY, PRO, SER 244 55 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR,TRP, TYR, VAL 245 56 ALA, ASN, CYS, GLY, SER, THR, VAL 246 57 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TRP, TYR, VAL 247 58 ALA, ARG, ASP, CYS, GLY, ILE, LYS, MET, PRO, SER248 59 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE,SER, THR, TRP, TYR, VAL 249 60 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,ILE, LEU, LYS, MET, PRO, SER, THR 250 61 ALA, ASN, ASP, CYS, GLN, GLU,GLY, ILE, SER, THR, VAL 251 62 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 252 63 ASN, ASP, GLY,SER 253 64 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE,SER, THR, TRP 254 65 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE,LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 255 66 ALA, ARG, ASN, ASP,CYS, MET, SER, THR 256 67 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS,ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 257 68 ALA, ARG, ASN,CYS, GLN, GLU, GLY, ILE, LYS, MET, PRO, SER, THR, VAL 258 69 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TRP, TYR, VAL 259 70 GLY 260 71 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 261 72 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR,TRP, TYR, VAL 262 73 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE,LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 263 74 GLY 264 75 ALA, ASN,ASP, CYS, GLN, GLU, GLY, PRO, SER, THR, TRP 265 76 ALA, ASN, ASP, CYS,GLN, GLU, GLY, SER, THR, VAL 266 77 ALA, CYS, GLY, SER 267 78 GLU 268 79ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 269 80 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 270 81 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, SER, THR, VAL 271 82 ASP 272 83ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, MET, SER, THR, VAL 273 84 ALA,ASN, ASP, CYS, GLN, GLU, GLY, SER, THR 274 85 ALA, ASN, ASP, CYS, GLY,ILE, SER, THR, VAL 275 86 ALA, CYS, GLY, SER 276 87 GLY 277 88 ALA, GLY,SER 278 89 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, MET, SER, THR,VAL 279 90 ALA, GLY, SER 280 91 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS,MET, PHE, PRO, SER, TRP, TYR 281 92 GLY 282 93 ALA, GLY, SER 283 94 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER,THR, TRP, TYR, VAL 284 95 CYS, HIS, ILE, LEU, MET, PHE, THR, TYR, VAL285 96 ALA, GLY, SER 286 97 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 28798 ALA, ASN, ASP, CYS, GLN, HIS, LEU, PHE, SER, TYR 288 99 HIS, PHE 289100 ALA, GLY, SER 290 101 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS,ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 291 102 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, PRO, SER, THR,TRP, TYR, VAL, 292 103 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU,LYS, MET, PHE, SER, THR, TRP, TYR, VAL 293 104 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, VAL 294 105 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO,SER, THR, TRP, TYR, VAL 295 106 GLY 296 107 ALA, ASN, ASP, CYS, GLN,GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, VAL 297 108 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 298109 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, SER, THR, TRP, TYR, VAL, 299 110 ALA, GLY, SER 300 111 ALA, ASN,ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 301 112 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, PHE, SER, THR,VAL 302 113 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, SER,THR, TRP, VAL 303 114 ALA, GLY, SER 304 115 ALA, ASN, ASP, CYS, GLN,GLU, GLY, ILE, LEU, SER, THR, VAL 305 116 ALA, ARG, ASN, ASP, CYS, GLN,GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 306 117 ALA,ASN, ASP, SER 307 118 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE,LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 308 119 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP,TYR, VAL, 309 120 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS,MET, PHE, SER, THR, TRP, TYR 310 121 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 311 122 ALA,CYS, GLY, PRO, SER 312 123 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS,LYS, MET, PHE, SER, THR, TRP, TYR 313 124 ALA, CYS, GLY, ILE, SER, THR,VAL 314 125 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR, VAL 315126 ALA, CYS, GLY, SER, THR 316 127 ALA, ASN, ASP, CYS, GLN, GLU, GLY,ILE, LEU, MET, SER, THR, VAL 317 128 ALA, ASN, CYS, GLY, SER, THR, VAL318 129 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL 319 130 ALA, ASN, ASP, CYS, GLN, GLY, HIS, MET,SER, THR 320 131 ALA, ARG, ASN, ASP, CYS, GLN, GLY, LYS, MET, SER 321132 ALA, CYS, GLY, PRO, SER 322 133 ALA, ASP, CYS, GLN, GLU, GLY, LEU,SER 323 134 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS,MET, PHE, PRO, SER, THR, TRP, VAL, 324 135 ALA, ARG, ASN, ASP, CYS, GLN,GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 325 136ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, TRP,TYR 326 137 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET,PHE, SER, THR, TRP, TYR 327 138 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 328 139 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER,THR, TRP, TYR, VAL, 329 140 ALA, ASP, CYS, GLY, SER 330 141 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 331 142ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL, 332 143 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 333 144 ALA, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, VAL 334 145ALA, ARG, ASN, ASP, CYS, GLU, GLY, MET, SER, THR, VAL 335 146 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TRP, TYR, VAL, 336 147 ALA, ARG, CYS, GLN, GLU, GLY, MET, SER 337 148ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER,THR, TRP, TYR, VAL 338 149 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS,ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 339 150 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP,TYR, VAL, 340 151 ALA, ASN, ASP, GLY, SER 341 152 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, SER, THR, VAL 342 153 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TRP, TYR, VAL, 343 154 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE,LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 344 155 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 345 156ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TYR, VAL 346 157 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS,MET, PHE, SER, THR 347 158 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS,MET, PRO, SER, THR, VAL 348 159 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU,SER, THR, VAL 349 160 ALA, CYS, GLY, SER, THR 350 161 ALA, ASN, ASP,CYS, GLY, SER, THR 351 162 ALA, GLY, SER 352 163 GLY 353 164 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, SER, THR, VAL 354 165 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR,TRP, TYR 355 166 GLY 356 167 ALA, GLY, SER 357 168 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, ILE, MET, SER, THR, VAL 358 169 ALA, GLY, SER 359170 ASN, GLY 360 171 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU,LYS, MET, SER, THR, VAL 361 172 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 362 173 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TRP, TYR, VAL, 363 174 ASN, ASP, GLY, SER 364 175 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 365 176ALA, ARG, ASN, ASP, CYS, GLY, HIS, MET, PHE, SER, THR, TRP, TYR 366 177ALA, ASN, ASP, CYS, HIS, LYS, SER 367 178 ALA, ASP, CYS, GLY, SER, THR,VAL 368 179 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE,SER, THR, TRP, TYR 369 180 ALA, CYS, HIS, PHE, SER, TYR 370 181 ALA,ASP, CYS, GLY, PRO, SER 371 182 ALA, GLY, SER 372 183 ALA, CYS, GLY, SER373 184 ALA, GLY, SER 374 185 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PRO, SER, THR, TRP, VAL 375 186 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR,VAL 376 187 ALA, ASN, ASP, CYS, GLY, HIS, ILE, LEU, SER, THR, VAL 377188 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,SER, THR, VAL 378 189 ALA, GLY, SER 379 190 ALA, ASP, CYS, GLY, SER, THR380 191 GLY 381 192 GLY 382 193 ALA, CYS, GLY, SER, THR 383 194 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 384195 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, SER, THR 385 196 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, TRP,VAL 386 197 ALA, CYS, GLY, MET, SER, THR 387 198 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR,VAL, 388 199 ASN, ASP, GLY, LYS, SER 389 200 GLY 390 201 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP,TYR, VAL, 391 202 ALA, ASN, ASP, CYS, GLN, GLY, ILE, MET, PRO, SER, THR,VAL 392 203 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET,PHE, SER, THR, TRP, TYR 393 204 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 394 205 ALA, CYS, GLN, GLU,GLY, SER, THR 395 206 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS,MET, SER, THR, VAL 396 207 ALA, CYS, GLY, SER, THR, VAL 397 208 ALA,CYS, PHE, TRP, TYR 398 209 ARG, ASN, ASP, CYS, GLN, MET, SER 399 210ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, LYS, MET, SER 400 211 GLY401 212 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, PRO, SER, THR, TRP, TYR, VAL 402 213 ALA, ARG, ASN, ASP, CYS, GLN,GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 403 214GLY 404 215 GLY 405 216 ALA, GLY, SER 406 217 ALA, CYS, GLY, SER, THR407 218 GLY 408 219 GLY 409 220 GLY 410 221 ALA, ASN, CYS, GLY, ILE,SER, THR, VAL 411 222 ALA, GLY, SER 412 223 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, VAL 413 224 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, PHE, SER, THR, TYR, VAL 414225 ALA, ASN, CYS, GLN, GLU, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TYR, VAL 415 226 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU,LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 416 227 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, PRO, SER, THR, VAL 417 228 ALA,CYS, GLN, GLU, GLY, MET, PRO, SER, THR 418 229 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR,VAL 419 230 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, PHE, SER, TRP,TYR 420 231 GLN, GLU 421 232 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 422 233 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER,THR, TRP, TYR, VAL 423 234 ALA, GLY, SER 424 235 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR,VAL, 425 236 ALA, CYS, GLY, PRO, SER, THR, VAL 426 237 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR,TRP, TYR, VAL 427 238 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS,MET, PHE, PRO, SER, THR, TRP, TYR, VAL 428 239 ALA, ASN, ASP, CYS, GLN,GLU, GLY, SER, THR, VAL 429 240 ALA, ASN, ASP, CYS, GLY, SER 430 241ALA, ASN, ASP, CYS, GLY, SER, THR 431 242 ALA, ARG, ASN, ASP, CYS, GLN,GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 432243 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, PRO, SER, THR, TRP, TYR, VAL 433 244 ALA, ARG, ASN, ASP, CYS, GLN,GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 434 245ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,PRO, SER, THR, TRP, TYR, VAL 435 246 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 436 247GLY 437 248 ALA, ARG, ASN, CYS, GLN, SER, THR 438 249 GLY 439 250 ALA,ASN, ASP, CYS, GLN, GLU, GLY, ILE, MET, SER, THR, VAL 440 251 ALA, GLY,PRO, SER 441 252 ASP 442 253 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU,MET, SER, THR 443 254 ALA, GLY, SER 444 255 ALA, GLY 445 256 ALA, ASN,ASP, CYS, GLY, SER 446 257 ALA, GLY, SER 447 258 ALA, ASN, ASP, CYS,GLY, SER, THR 448 259 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE,LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 449 260 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP,TYR, VAL, 450 261 ALA, ASN, ASP, CYS, GLY, HIS, SER, THR 451 262 GLY 452263 ALA, ASN, CYS, GLN, HIS, ILE, LEU, PHE, SER, THR, TYR, VAL 453 264ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL, 454 265 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL455 266 ALA, ARG, ASN, ASP, CYS, GLU, GLY, HIS, ILE, MET, PHE, SER, THR,TRP, TYR, VAL 456 267 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU,SER, THR, VAL 457 268 ALA, ASN, ASP, CYS, GLY, ILE, MET, SER, THR, TRP,VAL 458 269 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LYS, MET, SER 459270 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, SER, THR, TRP, TYR, VAL, 460 271 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TYR, VAL, 461 272 ALA,ASN, ASP, CYS, GLN, GLY, HIS, LYS, MET, SER, THR 462 273 ALA, ARG, ASN,ASP, CYS, GLN, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 463274 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, SER, THR, TRP, TYR, VAL, 464 275 GLY 465 276 GLY 466 277 ALA, ASN,ASP, CYS, GLY, SER, THR 467 278 SER 468 279 ALA, ASP, CYS, GLY, SER 469280 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 470 281 ALA, GLY, SER 471 282ALA, CYS, GLY, PRO, SER 472 283 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS,LEU, MET, SER, THR, VAL 473 284 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS,ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 474 285 ALA, GLY, SER475 286 ALA, GLY, SER 476 287 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, LEU, LYS, MET, SER, THR, VAL 477 288 ALA, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LYS, MET, SER, THR, VAL 478 289 ALA, GLY, SER 479 290ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER,THR, TRP, TYR 480 291 ALA, ARG, ASN, ASP, CYS, GLU, GLY, ILE, LEU, LYS,MET, SER, THR, VAL 481 292 ALA, ASN, ASP, CYS, GLN, GLU, GLY, MET, SER482 293 ALA, GLN, GLU, HIS, LYS, THR 483 294 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, TRP, TYR 484 295 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 485 296 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER,TRP, TYR, VAL 486 297 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE,LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 487 298 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PRO, SER, THR, TRP, VAL 488 299ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR,VAL 489 300 GLY 490 301 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS,LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 491 302 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR,VAL 492 303 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS,MET, PRO, SER, THR, VAL 493 304 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 494 305 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER,THR, TRP, TYR, VAL 495 306 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU,MET, SER, THR 496 307 ALA, HIS, PHE, SER, THR, TYR 497 308 ALA, ARG,ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR,TRP, TYR 498 309 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER,THR 499 310 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET,PHE, PRO, SER, THR, TRP, TYR 500 311 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 501 312ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL 502 313 ALA, ASN, ASP, CYS, GLY, ILE, MET, SER,THR, VAL 503 314 ALA, ASN, ASP, CYS, HIS, LEU, MET, PHE, SER, THR, TYR,VAL 504 315 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET,PHE, SER, THR, TRP, TYR, VAL 505 316 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, TRP, VAL 506 317 ALA, ASN,ASP, CYS, GLN, GLY, ILE, SER, THR, VAL 507 318 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 508 319ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,SER, THR, TRP, TYR, VAL 509 320 GLY 510 321 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, HIS, LYS, MET, SER, THR, TRP, TYR 511 322 ALA, ASN, ASP,CYS, GLY, SER 512 323 ALA, ASN, ASP, CYS 513 324 ALA, ASN, ASP, CYS,GLN, GLU, GLY, ILE, LYS, MET, SER, THR, VAL 514 325 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 515326 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, SER 516 327ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE,PRO, SER, THR, TRP, TYR, VAL 517 328 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 518 329 ALA,ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR,TRP, TYR 519 330 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU,LYS, MET, PHE, SER, THR, TRP, TYR, VAL 520 331 HIS, PHE, THR, TRP, TYR521 332 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, SER, THR, TYR, VAL 522 333 ALA, GLY, SER 523 334 CYS, GLY, HIS,LYS, MET, PHE, SER, TYR 524 335 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY,HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 525 336 GLY526 337 HIS, PHE, TRP 527 338 ALA, ASN, ASP, CYS, SER 528 339 ALA, GLY,PRO, SER 529 340 ALA, ASP, CYS, GLY, SER, THR 530 341 ALA, ASN, CYS,GLY, SER, THR, VAL 531 342 GLY 532 343 ALA, ARG, ASN, ASP, CYS, GLN,GLU, GLY, LEU, LYS, MET, SER 533 344 GLY 534 345 ALA, CYS, GLY, SER, THR535 346 ALA, CYS, GLY, PRO, SER, THR 536 347 ALA, ARG, ASN, ASP, CYS,GLN, GLU, GLY, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 537 348 GLY538 349 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET,PHE, SER, THR, TRP, TYR, VAL 539 350 ALA, ARG, ASN, ASP, CYS, GLN, GLU,GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 540 351 ALA, ASN, ASP,CYS, GLN, GLU, GLY, LEU, LYS, SER, THR, VAL 541 352 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, LEU, LYS, MET, SER, THR 542 353 ALA, ARG, ASN, ASP,CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR,VAL 543 354 ALA, ARG, CYS, GLN, GLU, GLY, MET, SER, THR 544 355 ALA,ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER, THR 545 356 ALA, ARG, ASN,ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP 546357 Any residue 547 358 Any residue 548 359 Any residue 549 360 Anyresidue 550 361 Any residue 551 362 Any residue 552 363 Any residue 553364 Any residue

In a further embodiment, the polypeptides of the invention comprise orconsist of the amino acid sequence selected from the group consisting ofSEQ ID NOs: 2-42, 44-60 and 72-112, and 114-130 and 150-155, shownbelow. These polypeptides have increased activity relative to Kuma010,as shown in the examples that follow, or provide for improved productionof the polypeptides. In one embodiment, the polypeptides comprise orconsist of the amino acid sequence selected from the group consisting ofSEQ ID NOs: 2-42, 55-60 and 72-112, and 125-130 and 150-155; thesepolypeptides all show improved activity to Kuma010.

The N-terminal domain is in bold font, and changes relative to Kuma 011are noted next to the polypeptide name. In all cases, the polypeptidesdescribed below may further comprise a histidine tag at the C-terminus.Any suitable histidine tag can be used; Any suitable histidine tag canbe used; in one embodiment the tag is linked to a TEV protease cut sit(ENLYFQS) (SEQ ID NO: 149) to allow for its efficient removal with TEVprotease after purification, for example, the tag may comprise orconsist of the amino acid sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO:139). In another embodiment, a cleavable histidine tag may beincorporated at the C-terminus, comprising the amino acid sequenceX_(N)PQ(L/Q)PX_(N)HHHHHH (SEQ ID NO: 131), wherein X_(N) is an linker ofbetween 1-25 amino acid residues. In one non-limiting example, thecleavable histidine tag may comprise the amino acid sequenceGSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 132).

Kuma011-K262E MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 2 (unprocessed), SEQ ID NO: 72(processed)) Kuma011-V268AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEAELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 3 (unprocessed), SEQ ID NO: 73(processed)) Kuma011-V268SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGESELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 4 (unprocessed), SEQ ID NO: 74(processed)) Kuma011-V268TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGETELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 5 (unprocessed), SEQ ID NO: 75(processed)) Kuma011-E269LMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVLLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 6 (unprocessed), SEQ ID NO: 76(processed)) Kuma011-E269TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 7 (unprocessed), SEQ ID NO: 77(processed)) Kuma011-L270AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVEADIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 8 (unprocessed), SEQ ID NO: 78(processed)) Kuma011-L270TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVETDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 9 (unprocessed), SEQ ID NO: 79(processed)) Kuma011-L270VMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVEVDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 10 (unprocessed), SEQ ID NO: 80(processed)) Kuma011-G319AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWAGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 11 (unprocessed), SEQ ID NO: 81(processed)) Kuma011-S354AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDAGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 12 (unprocessed), SEQ ID NO: 82(processed)) Kuma011-S354EMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDEGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 13 (unprocessed), SEQ ID NO: 83(processed)) Kuma011-S354OMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 14 (unprocessed), SEQ ID NO: 84(processed)) Kuma011-S354RMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDRGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 15 (unprocessed), SEQ ID NO: 85(processed)) Kuma011-S354YMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDYGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 16 (unprocessed), SEQ ID NO: 86(processed)) Kuma011-G358NMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTNGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 17 (unprocessed), SEQ ID NO: 87(processed)) Kuma011-G358SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 18 (unprocessed), SEQ ID NO: 88(processed)) Kuma011-G358QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTQGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 19 (unprocessed), SEQ ID NO: 89(processed)) Kuma011-G358TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTTGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 20 (unprocessed), SEQ ID NO: 90(processed)) Kuma011-H368FMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 21 (unprocessed), SEQ ID NO: 91(processed)) Kuma011-H368QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVQFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 22 (unprocessed), SEQ ID NO: 92(processed)) Kuma011-D399QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 23 (unprocessed), SEQ ID NO: 93(processed)) Kuma011-D402SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPSGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 24 (unprocessed), SEQ ID NO: 94(processed)) Kuma011-D402QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPQGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 25 (unprocessed), SEQ ID NO: 95(processed)) Kuma011-T406SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPQGGASGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 26 (unprocessed), SEQ ID NO: 96(processed)) Kuma011-N424KMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPQGGASGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 27 (unprocessed), SEQ ID NO: 97(processed)) Kuma011-A449EMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPETGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 28 (unprocessed), SEQ ID NO: 98(processed)) Kuma011-A449QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 29 (unprocessed), SEQ ID NO: 99(processed)) Kuma011-I456VMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVVDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 30 (unprocessed), SEQ ID NO: 100(processed)) Kuma011-T461RMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEARVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 31 (unprocessed), SEQ ID NO: 101(processed)) Kuma011-I463AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVAGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 32 (unprocessed), SEQ ID NO: 102(processed)) Kuma011-I463LMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVLGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 33 (unprocessed), SEQ ID NO: 103(processed)) Kuma011-I463MMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVMGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 34 (unprocessed), SEQ ID NO: 104(processed)) Kuma011-I463QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVQGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 35 (unprocessed), SEQ ID NO: 105(processed)) Kuma011-I463RMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVRGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 36 (unprocessed), SEQ ID NO: 106(processed)) Kuma011-I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 37 (unprocessed), SEQ ID NO: 107(processed)) Kuma011-I463VMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVVGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 38 (unprocessed), SEQ ID NO: 108(processed)) Kuma023: G368S; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 39 (unprocessed), SEQ ID NO: 109(processed))

Kuma 020 as referenced in the examples is the Kuma023 polypeptide thatincludes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH(SEQ ID NO: 139).

Kuma021: G368S; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 154 (unprocessed), SEQ ID NO: 155 (processed)) Kuma031: K262E;E269T; S354Q; G358S; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 40 (unprocessed), SEQ ID NO: 110 (processed))

Kuma 030 as referenced in the examples is the Kuma031 polypeptide thatincludes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH(SEQ ID NO: 139).

Kuma041: S319A; H368F; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWAGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 41 (unprocessed), SEQ ID NO: 111 (processed))

Kuma 040 as referenced in the examples is the Kuma041 polypeptide thatincludes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH(SEQ ID NO: 139).

Kuma051: K262E; E269T; L270V; S354Q; G358S; D399Q; A449QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVTVDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 42 (unprocessed), SEQ ID NO: 112 (processed))

Kuma 050 as referenced in the examples is the Kuma051 polypeptide thatincludes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH(SEQ ID NO: 139).

Kuma022: P171R; H172R; G368S; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 55 (unprocessed), SEQ ID NO: 125 (processed)) Kuma032: P171R;H172R; K262E; E269T; S354Q; G358S; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 56 (unprocessed), SEQ ID NO: 126 (processed)) Kuma042: P171R;H172R; S319A; H368F; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWAGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 57 (unprocessed), SEQ ID NO: 127 (processed)) Kuma052: P171R;H172R; K262E; E269T; L270V; S354Q; G358S; D399Q; A449QMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVTVDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 58 (unprocessed), SEQ ID NO: 128 (processed)) Kuma061: K262E;E269T; S354Q; G358S; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSMPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLG SPIGVRLLQALLPSASQPQP(SEQ ID NO: 150 (unprocessed), SEQ ID NO: 151 (processed))

Kuma 060 as referenced in the examples is the Kuma061 polypeptide thatincludes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH(SEQ ID NO: 139).

Kuma062: P171R; H172R; K262E; E269T; S354Q; G358S; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSMPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 59 (unprocessed), SEQ ID NO: 129 (processed)) Kuma071: S319A;H368F; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWAMPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 152 (unprocessed), SEQ ID NO: 153 (processed))

Kuma 070 as referenced in the examples is the Kuma071 polypeptide thatincludes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH(SEQ ID NO: 139).

Kuma072: P171R; H172R; S319A; H368F; D399Q; A449Q; I463TMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWAMPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 60 (unprocessed), SEQ ID NO: 130 (processed))

As described in Table 5, the changes made to Kuma010/011 havesignificant effect on the catalytic activity of the design proteins.Table 5 lists the effectiveness of individual mutations in catalyzingthe degradation of various gliadin peptide sequences. The examplesprovide further data regarding specific individual and combinationmutants.

TABLE 5 % Improvement on % Improvement Position A.A. relative PFPQPQLPYon (Full Position Kuma010 to (SEQ ID NO: PFPQPQQPF (SEQ Length)(Truncated) A.A. Kuma010/011 67) ID NO: 68) 221 32 E D, N, Q, H 105% ND262 73 K E 109% 110% 268 79 V A 107% 89% 268 79 V S 104% 83% 268 79 V T127% 105% 269 80 E L 113% 84% 269 80 E T 263% 191% 270 81 L A 203% 92%270 81 L T 307% 29% 270 81 L V 474% 61% 319 130 S A 154% 184% 354 165 SA 152% 140% 354 165 S E 124% 120% 354 165 S Q 145% 141% 354 165 S R 109%82% 354 165 S Y 46% 105% 358 169 G N 120% 99% 358 169 G S 331% 224% 358169 G Q 147% 149% 358 169 G T 283% 128% 368 179 H F 334% 104% 368 179 HQ 199% 195% 399 210 D Q 149% 208% 402 213 D S 94% 108% 402 213 D Q 164%111% 406 217 T S 84% 101% 424 235 N K 285% ND 449 260 A E 149% 208% 449260 A N 119% 118% 461 272 T R 120% 86% 463 274 I A 51% 234% 463 274 I L124% 22% 463 274 I M 123% 53% 463 274 I Q 129% 69% 463 274 I R 29% 110%463 274 I T 130% 239% 463 274 I V 256% 141%

Improved Production Mutations

Mutations that improve production may provide improvements in one ofthree categories: 1. Altering purification method; 2. increase in yield;and 3. decreasing the probability that enzymatic self-processing wouldoccur during purification, thereby simplifying analysis. Addition of aHis tag that is removable by the proteolytic activity of thepolypeptides disclosed herein falls into category 1; R105H mutantappears to improve yield by ˜2-fold, placing this mutation into category2; and mutations in positions 171-174 place these mutants into category3.

Kuma010 with Kuma010-Cleavable His TagMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQALLPSASQPQPGSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 43 (unprocessed),SEQ ID NO: 113 (processed)) Kuma011-R105HMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 44 (unprocessed), SEQ ID NO: 114 (processed)) Kuma011-P171AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARAHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 45 (unprocessed), SEQ ID NO: 115 (processed)) Kuma011-P171RMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 46 (unprocessed), SEQ ID NO: 116 (processed)) Kuma011-P171SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARSHFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGS PIGVRLLQALLPSASQPQP(SEQ ID NO: 47 (unprocessed), SEQ ID NO: 117 (processed)) Kuma011-H172AMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPAFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 48 (unprocessed), SEQ ID NO: 118 (processed)) Kuma011-H172RMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 49 (unprocessed), SEQ ID NO: 119 (processed)) Kuma011-H172SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPSFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 50 (unprocessed), SEQ ID NO: 120 (processed)) Kuma011-F173RMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHRRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 51 (unprocessed), SEQ ID NO: 121 (processed)) Kuma011-F173SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHSRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 52 (unprocessed), SEQ ID NO: 122 (processed)) Kuma011-R174SMSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARPHFSMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 53 (unprocessed), SEQ ID NO: 123 (processed)) Kuma012: P171R;H172R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQID NO: 54 (unprocessed), SEQ ID NO: 124 (processed))

As used throughout the present application, the term “polypeptide” isused in its broadest sense to refer to a sequence of subunit aminoacids, whether naturally occurring or of synthetic origin. Thepolypeptides of the invention may comprise L-amino acids, D-amino acids(which are resistant to L-amino acid-specific proteases in vivo), or acombination of D- and L-amino acids. The polypeptides described hereinmay be chemically synthesized or recombinantly expressed. Thepolypeptides may be linked to other compounds to promote an increasedhalf-life in vivo, such as by PEGylation, HESylation, PASylation, orglycosylation. Such linkage can be covalent or non-covalent as isunderstood by those of skill in the art. The polypeptides may be linkedto any other suitable linkers, including but not limited to any linkersthat can be used for purification or detection (such as FLAG or Histags).

In another aspect, the present invention provides isolated nucleic acidsencoding the polypeptide of any aspect or embodiment of the invention.The isolated nucleic acid sequence may comprise RNA or DNA. As usedherein, “isolated nucleic acids” are those that have been removed fromtheir normal surrounding nucleic acid sequences in the genome or in cDNAsequences. Such isolated nucleic acid sequences may comprise additionalsequences useful for promoting expression and/or purification of theencoded protein, including but not limited to polyA sequences, modifiedKozak sequences, and sequences encoding epitope tags, export signals,and secretory signals, nuclear localization signals, and plasma membranelocalization signals. It will be apparent to those of skill in the art,based on the teachings herein, what nucleic acid sequences will encodethe polypeptides of the invention.

In a further aspect, the present invention provides nucleic acidexpression vectors comprising the isolated nucleic acid of anyembodiment of the invention operatively linked to a suitable controlsequence. “Recombinant expression vector” includes vectors thatoperatively link a nucleic acid coding region or gene to any controlsequences capable of effecting expression of the gene product. “Controlsequences” operably linked to the nucleic acid sequences of theinvention are nucleic acid sequences capable of effecting the expressionof the nucleic acid molecules. The control sequences need not becontiguous with the nucleic acid sequences, so long as they function todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the nucleic acid sequences and the promoter sequence canstill be considered “operably linked” to the coding sequence. Other suchcontrol sequences include, but are not limited to, polyadenylationsignals, termination signals, and ribosome binding sites. Suchexpression vectors can be of any type known in the art, including butnot limited plasmid and viral-based expression vectors. The controlsequence used to drive expression of the disclosed nucleic acidsequences in a mammalian system may be constitutive (driven by any of avariety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive). The construction of expression vectors for use intransfecting prokaryotic cells is also well known in the art, and thuscan be accomplished via standard techniques. (See, for example,Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.). The expression vector must be replicable in the host organismseither as an episome or by integration into host chromosomal DNA. In apreferred embodiment, the expression vector comprises a plasmid.However, the invention is intended to include other expression vectorsthat serve equivalent functions, such as viral vectors.

In another aspect, the present invention provides recombinant host cellscomprising the nucleic acid expression vectors of the invention. Thehost cells can be either prokaryotic or eukaryotic. The cells can betransiently or stably transfected or transduced. Such transfection andtransduction of expression vectors into prokaryotic and eukaryotic cellscan be accomplished via any technique known in the art, including butnot limited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc.New York, N.Y.). A method of producing a polypeptide according to theinvention is an additional part of the invention. The method comprisesthe steps of (a) culturing a host according to this aspect of theinvention under conditions conducive to the expression of thepolypeptide, and (b) optionally, recovering the expressed polypeptide.The expressed polypeptide can be recovered from the cell free extract,cell pellet, or recovered from the culture medium. Methods to purifyrecombinantly expressed polypeptides are well known to the man skilledin the art.

In a further aspect, the present invention provides pharmaceuticalcompositions, comprising the polypeptide, nucleic acid, nucleic acidexpression vector, and/or the recombinant host cell of any aspect orembodiment of the invention, and a pharmaceutically acceptable carrier.The pharmaceutical compositions of the invention can be used, forexample, in the methods of the invention described below. Thepharmaceutical composition may comprise in addition to the polypeptides,nucleic acids, etc. of the invention (a) a lyoprotectant, (b) asurfactant: (c) a bulking agent; (d.) atonicity adjusting agent; (e)stabilizer; (f) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is aTris buffer, a histidine buffer, a phosphate buffer, a citrate buffer oran acetate buffer. The pharmaceutical composition may also include alyoprotectant, e.g. sucrose, sorbitol or trehalose. In certainembodiments, the pharmaceutical composition includes a preservative e.g.benzalkonium chloride, benzethonium, chlorohexidine, phenol,Jia-cresol., benzyl alcohol, methylparaben, propylparaben,chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate,thimerosal, benzoic acid, and various mixtures thereof. In otherembodiments, the pharmaceutical composition includes a bulking agent,like glycine. In yet other embodiments, the pharmaceutical compositionincludes a surfactant e.g., polysorbate-20, polysorbate-40,polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85,poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitanmonostearate, sorbitan monooleate, sorbitan trilaurate, sorbitantristearate, sorbitan trioleaste, or a combination thereof. Thepharmaceutical composition may also include a tonicity adjusting agent,e.g., a compound that renders the formulation substantially isotonic orisoosinotic with human blood. Exemplary tonicity adjusting agentsinclude sucrose, sorbitol, glycine, methionine, mannitol, dextrose,inositol, sodium chloride, arginine and arginine hydrochloride. In otherembodiments, the pharmaceutical composition additionally includes astabilizer, e.g., a molecule which, when combined with a protein ofinterest substantially prevents or reduces chemical and/or physicalinstability of the protein of interest in lyophilized or liquid form.Exemplary stabilizers include sucrose, sorbitol, glycine, inositol,sodium chloride, methionine, arginine, and arginine hydrochloride.

The polypeptides, nucleic acids, etc. of the invention may be the soleactive agent in the pharmaceutical composition, or the composition mayfurther comprise one or more other active agents suitable for anintended use.

The pharmaceutical compositions described herein generally comprise acombination of a compound described herein and a pharmaceuticallyacceptable carrier, diluent, or excipient. Such compositions aresubstantially free of non-pharmaceutically acceptable components, i.e.,contain amounts of non-pharmaceutically acceptable components lower thanpermitted by US regulatory requirements at the time of filing thisapplication. In some embodiments of this aspect, if the compound isdissolved or suspended in water, the composition further optionallycomprises an additional pharmaceutically acceptable carrier, diluent, orexcipient. In other embodiments, the pharmaceutical compositionsdescribed herein are solid pharmaceutical compositions (e.g., tablet,capsules, etc.).

The compositions described herein could also be provided as a dietarysupplement as described by the US regulatory agencies.

These compositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by any suitable route. In apreferred embodiment, the pharmaceutical compositions and formulationsare designed for oral administration. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable.

The pharmaceutical compositions can be in any suitable form, includingbut not limited to tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules, sterileinjectable solutions, and sterile packaged powders.

In another aspect, the present invention provides methods for treatingceliac sprue or non-celiac gluten sensitivity (NCGS), comprisingadministering to an individual with celiac sprue or NCGS an amounteffective to treat the celiac sprue or NCGS of one or more polypeptidesselected from the group consisting of the polypeptides of the of theinvention, or using one or more of these polypeptides to process foodfor consumption by individuals with celiac sprue or NCGS

The inventors of the present invention have discovered that thepolypeptides of the invention are capable of degrading proline (P)- andglutamine (Q)-rich components of gluten known as ‘gliadins’ believedresponsible for the bulk of the immune response in most celiac spruepatients. The polypeptides of the present invention show superioractivity in degrading peptides having a PQLP (SEQ ID NO: 65) or PQQP(SEQ ID NO: 66) motif (such as PFPQPQLPY (SEQ ID NO: 67) and/orPFPQPQQPF (SEQ ID NO: 68)), which are substrates representative ofgliadin) at pH 4 compared to Kuma010/011 and other polypeptidesdisclosed as useful for treating celiac sprue (WO2015/023728). Thus, thepolypeptides of the invention constitute significantly improvedtherapeutics for treating celiac sprue and NCGS.

Celiac sprue (also known as celiac disease or gluten intolerance) is ahighly prevalent disease in which dietary proteins found in wheat,barley, and rye products known as ‘glutens’ evoke an immune response inthe small intestine of genetically predisposed individuals. Theresulting inflammation can lead to the degradation of the villi of thesmall intestine, impeding the absorption of nutrients. Symptoms canappear in early childhood or later in life, and range widely inseverity, from diarrhea, fatigue, weight loss, abdominal pain, bloating,excessive gas, indigestion, constipation, abdominal distension,nausea/vomiting, anemia, bruising easily, depression, anxiety, growthdelay in children, hair loss, dermatitis, missed menstrual periods,mouth ulcers, muscle cramps, joint pain, nosebleeds, seizures, tinglingor numbness in hands or feet, delayed puberty, defects in tooth enamel,and neurological symptoms such as ataxia or paresthesia. There arecurrently no effective therapies for this lifelong disease except thetotal elimination of glutens from the diet. Although celiac sprueremains largely underdiagnosed, its' prevalence in the US and Europe isestimated at 0.5-1.0% of the population. In addition to celiac sprue, asignificant fraction of the population is thought to suffer from thecondition of non-celiac gluten sensitivity (NCGS), which is caused bythe ingestion of gluten but is mechanistically distinct from celiacdisease, though the symptoms are frequently indistinguishable from thoseof celiac sprue.

As used herein, “treating celiac sprue or NCGS” means accomplishing oneor more of the following: (a) reducing the severity of celiac sprue orNCGS; (b) limiting or preventing development of symptoms characteristicof celiac sprue or NCGS; (c) inhibiting worsening of symptomscharacteristic of celiac sprue or NCGS; (d) limiting or preventingrecurrence of celiac sprue or NCGS in patients that have previously hadthe disorder; (e) limiting or preventing recurrence of symptoms inpatients that were previously symptomatic for celiac sprue or NCGS; and(f) limiting development of celiac sprue or NCGS in a subject at risk ofdeveloping celiac sprue or NCGS, or not yet showing the clinical effectsof celiac sprue or NCGS.

The individual to be treated according to the methods of the inventionmay be any individual suffering from celiac sprue or NCGS, includinghuman subjects. The individual may be one already suffering fromsymptoms or one who is asymptomatic.

As used herein, an “amount effective” refers to an amount of thepolypeptide that is effective for treating celiac sprue. Thepolypeptides are typically formulated as a pharmaceutical composition,such as those disclosed above, and can be administered via any suitableroute, including orally, parentally, by inhalation spray, or topicallyin dosage unit formulations containing conventional pharmaceuticallyacceptable carriers, adjuvants, and vehicles. In a preferred embodiment,the pharmaceutical compositions and formulations are orallyadministered, such as by tablets, pills, lozenges, elixirs, suspensions,emulsions, solutions, or syrups.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). A suitable dosage rangemay, for instance, be 0.1 ug/kg-100 mg/kg body weight; alternatively, itmay be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10mg/kg body weight. The polypeptides can be delivered in a single bolus,or may be administered more than once (e.g., 2, 3, 4, 5, or more times)as determined by an attending physician.

EXAMPLE 1

Gliadin is highly enriched in proline (P) and glutamine (Q), whichrenders it recalcitrant to degradation by human digestive enzymes.PQ-rich peptide fragments derived from partial digestion of gliadin aredeamidated in the intestinal lumen, thereby allowing binding to HLA-DQ2or DQ8, and stimulation of a Th1 inflammatory response in people withCD³. The gliadin endopeptidase KUMAMAX™ (hereafter referred to asKuma011, or Kuma 010 if referring to Kuma011 including the C-terminalhistidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139)), whichdemonstrates stability and functionality in gastric conditions, waspreviously engineered to break down peptides containing the PQ dipeptidemotif. Based on the crystal structure of Kuma010 (PDB ID 4NE7), weredesigned the active site of Kuma010 selecting for mutations toincrease activity against immunogenic gliadin peptides. Designed mutantswere then screened for increased activity against the highly immunogenic33mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69)) and 26mer(FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70)) gliadin peptides^(6,7).These peptides harbor either the PQL or PQQ tripeptide motif,representative of all gliadin T cell epitopes shown to be toxic for thevast majority of celiac patients⁸. In this manner, the variant Kuma030was built. Kuma030 is 44-fold more active against peptides containingPQQ, and 11-fold more active against peptides containing PQL, thanKuma010.

Based on the molecular modeling, the putative S1′ peptide bindinginterface of Kuma010 consists of entirely hydrophobic residues, andshould therefore prefer hydrophobic residues such as leucine, and notpolar residues such as glutamine, at P1′. The S1′ binding pocket ofKuma030 introduces an isoleucine to threonine mutation (I463T), which ispredicted to provide a hydrogen bond with a P1′ glutamine, enabling thisenzyme to accommodate both leucine and glutamine in the S1′ subsite andthereby target both PQL and PQQ tripeptides. Kuma030 also incorporatessix additional mutations (K262E, E269T, S354Q, G358S, D399Q, A449Q) thatprovide enhanced catalytic efficiency on the 26mer and 33mer peptides.G358S is predicted to stabilize the loop containing an engineeredhistidine introduced in Kuma010 which is predicted to hydrogen bond tothe P1 glutamine residue. The remaining mutations are predicted tostabilize the protein structure as modeled.

While dozens of PQ-rich epitopes have been linked to CD, severalpeptides derived from gliadin (wheat), hordein (barley), and secalin(rye) have been shown to account for the vast majority of the immuneresponse in CD and have thus been classified as immunodominant⁸. Inwheat, these include the peptides W02-E07 (LQPFPQPQLPYPQPQ (SEQ ID NO:133)), W03-E07 (QPFPQPQQPFPWQP (SEQ ID NO: 134)), and the 33mer peptide,which contains the W02-E07 sequence^(6,9). These peptides harbor severalepitopes shown to be highly immunogenic⁹⁻¹¹. To evaluate the ability ofKuma030 to destroy these epitopes throughout gluten, purified wholegluten was incubated with Kuma030 under simulated gastric conditions (pH4.0 at 37° C. with 0.6 mg mL⁻¹ pepsin)¹². The gluten fraction remainingafter degradation was quantified using ELISA assays based on either theR5 or G12 antibodies recognizing the amino acid motifs QQPFP (SEQ ID NO:135) and QPQLPY (SEQ ID NO: 136), respectively, which encompass all ofthe immunodominant epitopes in the above peptides^(13,14). To comparethe activity of Kuma030 to that of published glutenases, we alsoexamined the glutenases EPB2 and SCPEP, which are currently beingpioneered at a 1:1 ratio as a combination enzyme therapeutic for CD¹⁵.The EPB2 and SCPEP enzymes generated in this work were verified to haveactivities consistent with that of published values^(16,17). Uponincubation with gluten, we observed a dose-dependent reduction in theQQPFP (SEQ ID NO: 135) or QPQLPY (SEQ ID NO: 136) load using eitherKuma030, Kuma010, or a 1:1 combination of EPB2 and SCPEP (FIG. 1A). At a1:25 w:w ratio of enzyme:gluten, EPB2 and SCPEP broke down 84.8% of thegluten present, consistent with previously published reports of thisenzyme combination^(18,19). Kuma030 was highly effective at eliminatingthese peptide epitopes throughout gluten, with a 1:400 w:w ratio ofenzyme:gluten being sufficient to reduce the immunogenic gluten presentby over 99.5% at 60 minutes, as quantified by both methods used (FIG.1A). Kuma030-dependent reduction in immunogenic gluten load is rapid,with >98% degradation achieved by 5 minutes at a w:w ratio of 1:25 (FIG.1B). Mass spectroscopy of the Kuma030 cleavage products revealed thatKuma030 cleaved each peptide after the PQ dipeptide motif in theimmunodominant epitopes from wheat (33mer, W02-E07, and W03-E07), andalso those from barley B08-E2E7 (PQQPIPQQPQPYPQQ (SEQ ID NO: 61)) andrye R11-E2E7 (QPFPQQPEQIIPQQP)⁹ (SEQ ID NO: 62) (FIG. 1C). While theseintact peptides are highly immunostimulatory, the peptide breakdownproducts are not predicted to stimulate the immune system, since Kuma030action results in elimination of the core 9mer epitope thought totrigger the immune response²⁰. The ability of Kuma030 to cleave thesepeptides containing either a PQL or a PQQ tripeptide motif, and toreduce the gliadin load as measured by both G12 and R5 antibodies, areconsistent with the hypothesis that Kuma030 can bind and cleave peptideswith either a leucine or a glutamine in the S1′ binding pocket.

The ability of Kuma030 to efficiently degrade immunogenic gliadinepitopes suggests that incubation of gliadin with Kuma030 might reduceits capacity to stimulate a T-cell mediated immune response. T cellassays utilizing cells derived from the intestinal biopsies of celiacpatients represent the gold standard for this evaluation. To directlyevaluate the hypothesis that incubation with Kuma030 would decrease oreliminate the immunostimulatory capacity of gliadin, we performed T cellassays in which cells were exposed to Kuma030-treated gliadin and theresulting T cell reaction was assessed. The highly gliadin-reactiveintestinal CD4⁺ T cell lines used in this study were previouslygenerated from intestinal mucosa and have been shown to react to adiversity of epitopes across different gliadin families²¹. Kuma030 andpepsin were incubated with purified wheat gliadin inlaboratory-simulated gastric conditions for 60 minutes. In order tomimic transit into the intestinal compartment, the pH levels of thesamples were then increased, and the samples were treated withchymotrypsin and deamidated with TG2 to unmask the immunogenic epitopes.The resulting gliadin samples were presented to T cell lines, andstimulation was assessed by measuring IFN-γ production (FIG. 2A-E) and Tcell proliferation (FIG. 3A-D). Exposure of T cell lines topepsin-treated gliadin resulted in stimulation of production of IFN-γ,and co-treatment of gliadin with pepsin and Kuma030 reduced thisresponse, eliminating it at higher concentrations. The decreasesobserved in IFN-γ production were not due to Kuma030 toxicity.Importantly, Kuma030 eliminated the T cell response to gliadin in everyT cell line tested regardless of T cell epitope specificity, indicatingthat Kuma030 is effective against all epitopes recognized by the T celllines used in this assay. Since these epitopes span the three majorgliadin families: α-, ω-, and γ-gliadin, this suggests that Kuma030 iscapable of degrading immunogenic epitopes within all relevant regions ofgliadin.

The experiments above demonstrate the ability of Kuma030 to degradeimmunogenic gliadin epitopes in the context of purified whole gluten orgliadin. However, to assess practical application, it is important toevaluate Kuma030 effectiveness in physiologically relevant food andbeverage matrices. To assess the activity of Kuma030 in gastricdigestion scenarios, we tested the ability of Kuma030 to break downgluten in an acidified bread slurry and in a wheat beer. Whole-wheatbread was mashed in artificial saliva to simulate mastication at a breadconcentration representative of that in the stomach after ingestion ofone slice of bread. The mixture was then acidified by the addition ofHCl and pepsin, and glutenases at various concentrations were added. Theamount of gluten remaining was then quantified after 30 minutes ofdigestion, which represents the average lag time of food in the stomachbefore the commencement of ingesta release into the duodenum through thepyloric opening²². At the highest concentration of glutenase tested(1000 μg mL⁻¹), treatment with EPB2 and SCPEP resulted in84.4% glutendegradation (FIG. 4A). This is comparable to published results from aPhase I study showing that EPB2 and SCPEP eliminate 70-79% of gluten ina test meal at a 1:10 enzyme:gluten ratio in the human stomach after a30 minute incubation time²³. At a concentration of 62.5 μg mL⁻¹ (a 1:160w:w ratio), Kuma030 reduced the level of gluten in the bread to below 20ppm (the FDA threshold for “gluten free” labeling). Finally, thegluten-degrading ability of Kuma030 was tested directly in a wheat beer,since wheat beers demonstrate comparatively high gluten levels²⁴. Beerwas incubated with Kuma030 at 37° C. or 4° C. at two enzymeconcentrations. Samples were taken at various timepoints and theconcentration of remaining gluten was quantified. We found thatincubation of beer, which demonstrated a gluten concentration of ˜764ppm, with Kuma030 decreased the gluten level to below 20 ppm by only 5minutes (FIG. 4B). The rapidity of this effect is particularly importantsince liquids empty through the stomach considerably faster thansolids²⁵. Surprisingly, Kuma030 significantly reduced the gluten load ofwheat beer even at 4° C., as incubation with the Kuma030 at 700 μg mL⁻¹reduced the gluten levels to below 20 ppm by 5 minutes.

At this time, the only therapy for celiac disease is a lifelong strictgluten free diet. Oral enzyme therapy has been considered an attractivetreatment option for CD since the identification of PQ-rich immunogenicgliadin epitopes that stimulate the immune response³. A usefulcharacteristic of any oral enzyme therapeutic for CD is the ability tobreak down immunogenic peptides in gastric conditions, since theinflammatory immune response to gliadin occurs immediately upon enteringthe intestine²⁶. Gluten challenge studies in CD patients have shown thatthe ingested gluten load must be kept at 10 mg or less in order toprevent intestinal damage^(27,28). Indeed, the FDA currently mandatesthat any food labeled as “gluten free” must demonstrate less than 20 ppmgluten, since strict adherence to this standard is predicted to resultin a daily ingestion of 10 mg or less. Thus, the accidental ingestion of1 g of gluten (approximately the amount of gluten present in a crouton)must be reduced by 99% or greater in the gastric compartment in order toprevent the intestinal damage and symptoms that would arise from glutenexposure. There is therefore a clear need for glutenases that canrapidly destroy immunogenic gliadin epitopes in gastric conditions. In abread slurry model representing ingestion of 4 g of gluten, Kuma030 wasfound to degrade >99.8% of the gliadin load in 30 minutes at a 1:160 w:wratio. Additionally Kuma030 specifically destroys peptides with the PQdipeptide motif, which is commonly found throughout the immunogenicregions of gluten. Indeed, Kuma030 is capable of degrading allimmunodominant peptides tested, and gliadin treated with Kuma030 failedto stimulate IFN-γ production by all T cell lines tested, which issignificant since CD patients demonstrate a myriad of responses todifferent immunogenic epitopes.

REFERENCES FOR EXAMPLE 1

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EXAMPLE 2

Computationally designed enzymes were then produced and tested for theirability to break down immunogenic gliadin peptides. Mutations that wereshown to improve the ability of the enzyme to target relevant peptideswere then combined and tested in an iterative process to furtherincrease activity. More recently, design has been extended to the S1′binding pocket to prefer either L or Q amino acids. This engineeringeffort has greatly increased activity on peptides containing a PQL orPQQ tripeptide, which are located within the core epitope of virtuallyall immunogenic gliadin peptides.

Several Kuma010 variants are used. The specific mutational differences,and their relative effects on activity, are listed in Table 6 below.

TABLE 6 Activity Improvement Variant Background^(a) Mutation^(b) overBackground^(c) Kuma010 Kumamolisin-As V119D 116x S262K N291D D293T G319SD358G D368H Kuma020 Kuma010 G358S 7-19x (PQL) I463T 15-35x (PQQ) Kuma021Kuma020 D399Q 1-2x (PQL) A449Q 1.2-2.5x (PQQ) Kuma030 Kuma021 K262E1.2-2x (PQL) E267T 1.2-2x (PQQ) S354Q Kuma031 Kuma030GSTENLYFQSGALEHHHHHH No change (PQL); (SEQ ID NO: 139) production mutantDeleted from C-terminus Kuma032 Kuma031 P171R No change (PQL)); H172Rproduction mutant Kuma040 Kuma010 S319A 35-50x (PQL) H368F 30-40x (PQQ);D399Q Kuma030 is 2-fold A449Q more active on I463T PQQ, and 2-fold lessactive on PQL, than Kuma040 Kuma041 Kuma040 GSTENLYFQSGALEHHHHHH1.0-1.2x (PQL) (SEQ ID NO: 139) Deleted from C-terminus Kuma042 Kuma041P171R 1.2-1.5x (PQL) H172R Kuma050 Kuma021 K262E 1.5-2x (PQL) E267T0.01-0.2x (PQQ) S354Q L270V T463I Kuma060 Kuma030 G320M In bread assays.Kuma060 is about twice as active as Kuma030 Kuma061 Kuma060GSTENLYFQSGALEHHHHHH about same (SEQ ID NO: 139) activity as Deletedfrom C-terminus Kuma060. Kuma062 Kuma061 P171R ND^(d); about same H172Ractivity as Kuma060. Kuma070 Kuma040 G320M 2-3x (PQL) 2-4x (PQQ) Kuma071Kuma070 GSTENLYFQSGALEHHHHHH ND^(d;) about 0.2X as (SEQ ID NO: 139)active as Kuma Deleted from C-terminus 070 Kuma072 Kuma070GSTENLYFQSGALEHHHHHH about 0.7X as (SEQ ID NO: 139) active as KumaDeleted from C-terminus 070; P171R H172R ^(a)The specific Kuma010variant that served as a background upon which the listed mutations weremade. ^(b)Mutational positions are denoted relative to the full-lengthKuma010 enzyme. ^(c)Activity Improvement was calculated for each variantas a fold increase in activity relative to that of the“background” enzyme, the template enzyme used to make that variant.Activity was measured on one or more of the following substrates: afluorescent-labeled PQPQLP (SEQ ID NO: 156) substrate, the 33mer⁵(contains PQL) or 26mer⁶ (contains PQQ) peptides, or DQ2.5-glia-α1a(contains PQL) or DQ2.5-glia-ω1 (contains PQQ)⁷. It is important to notethat since hundreds of Kuma010 variants were testes, it would have beenimpractical to obtain kinetic constants for each mutant, thus the foldimprovement numbers shown here are estimates instead of definitivenumbers. The fold improvement numbers presented here are calculated fromthe amount of peptide degradation product detected in the degradationassay by LC-MS. ^(d)ND: not determined. The activities of leads Kuma060,Kuma061, and Kuma062; and Kuma070, Kuma071, and Kuma072; were compareddirectly to each other in bread or meal degradation assays instead of byassessment of individual peptide degradation in order to confirm that nodecrease in enzymatic activity occurred upon loss of the His tag. Theability of Kuma070 to break down PQL- and PQQ-containing peptides wascompared to Kuma040 as discussed below.

Kuma020, Kuma021, and Kuma030

The active site of Kuma010 underwent further design effort to improveactivity against substrates containing either PQL or PQQ tripeptides.Engineering efforts identified the mutations G358S and I463T asimportant contributors to increased activity. The G358S mutation was arefinement to a previous mutation made at this site in Kuma010. TheI463T mutation eliminated the steric hindrance encountered in the P1′binding pocket when targeting PQL tripeptide motifs, and introduced anew predicted hydrogen bond when PQQ was the substrate⁴. The Kuma010variant harboring these two mutations demonstrated a very largeimprovement over the original Kuma010 and was named Kuma020.

Additional mutations were made to this Kuma020 enzyme. D399Q and A449Qwere mutations that were located outside of the active site, and so arenot predicted to affect binding to the substrate. Instead, these twomutations resulted in new predicted intramolecular hydrogen bonds andwere thus predicted to stabilize the enzyme. The resulting variant,Kuma021, showed a further increase in activity.

Three other refinements contributed to the generation of Kuma030 fromKuma021. Kuma030 is described in detail above.

Kuma040 and Kuma050

As an alternative to the mutations listed above, a different set ofmutations on the Kuma010 background, S319A and H368F, led to a differentactive site architecture than the one found in Kuma030. Together thesemutations, along with D399Q, A449Q, and I463T, (mutations that alsoincreased activity in Kuma030) make up the Kuma040 variant. Kuma041,Kuma042, Kuma070, Kuma071, and Kuma072 have Kuma040-like active sites,while Kuma031, Kuma032, Kuma060, Kuma061, and Kuma062 have Kuma030-likeactive sites.

Kuma050 is a Kuma010 variant built on the Kuma021 background with anactive site architecture that has more in common with Kuma030 thanKuma040. However, Kuma050 lacks the I463T mutation, and instead harborsa L270V mutation which is predicted to increase its activity againstPQL-containing peptides but is predicted to hinder glutamine fromaccessing the P1′ binding pocket, thereby decreasing activity onPQQ-containing substrates. Accordingly, Kuma050 specificallydemonstrates a high level of activity against substrates containing PQL,but not against substrates containing PQQ. The specificity profile ofKuma050 was desired due to the fact that several studies have indicatedthat the immunodominant 33mer peptide from α-gliadin, which containsseveral PQL motifs and no PQQ motifs, may be the peptide responsible forthe vast majority of the disease in a subset of patients.

The activities of Kuma010, Kuma020, Kuma030, Kuma040, and Kuma050 areshown below for the highly immunogenic 33mer peptide of α-gliadin(LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69)) and the 26merpeptide of γ-gliadin (FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70)), whichrepresent PQL- and PQQ-containing peptides, respectively. The appearanceof breakdown products over time (LPYPQPQPF (SEQ ID NO: 137) for 33merpeptide; FLQPQ (SEQ ID NO: 138) for 26mer peptide) are shown in FIGS.5A-B.

In both cases, Kuma030 was the dominant enzyme tested, though Kuma040demonstrated activity almost as potent as Kuma030 especially against the26mer peptide. As expected, Kuma050 demonstrated good activity on the33mer peptide, but very poor activity on the 26mer peptide. Compared toKuma030 and Kuma040, Kuma020 demonstrated a moderate amount of activityon both peptides, consistent with it being a design intermediate asshown above. All variants demonstrate a marked improvement on theoriginal Kuma010 enzyme.

Kuma060 and Kuma070

An additional designed mutation was the mutation G320M. This mutationdid not appear particularly promising as it was not predicted to greatlyimprove activity, and indeed, this residue, while in the active site,does not appear to make direct contact with the gliadin substrate.However, mutation at this position improved activity by 2-4 fold on bothsubstrates. This could be due to slight changes in the Kuma010 backboneincurred by incorporation of the methionine, rendering it into aconfirmation that is even more favorable for catalysis. The G320Mmutation was incorporated into the Kuma030 and Kuma040 backgrounds togenerate enzymes Kuma060 and Kuma070, respectively.

FIGS. 6A-B show the improvement in activity attributed to the G320Mmutation on the Kuma040 background in degradation of both immunogenicepitopes DQ2.5-glia-α1a (contains PQL), and DQ2.5-glia-ω1 (containsPQQ).

The KumaXX1 and KumaXX2 Series

With the exception of Kuma021, Kuma010 variants labeled KumaXX1 (ex.Kuma031) correspond to the KumaXX0 variant (ex. Kuma030) in which theC-terminal TEV protease cut site and 6×His tag have been geneticallydeleted (GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139)). This tag, which wasoriginally added to the Kumamolisin-As enzyme for high-throughput, easypurification of Kuma010 variants, was removed in certain Kuma010 leadvariants as the 6× His tag is not preferable on biologicpharmaceuticals. In general, the removal of this tag did not affect theactivity of the enzyme, though His tag removal did appear to result in aslight decrease in the ability of the Kuma070 enzyme (but not theKuma060 enzyme) to degrade gliadin in a gastric digestion of whole wheatbread.

Kuma010 variants labeled KumaXX2 (ex Kuma032) also lack the His tag, andcontain the following additional mutations: P171R and H172R. Thesemutations don't affect activity of the enzyme, but were incorporated tosimplify the purification process. These mutations were introduced intothe propeptide domain in the N-terminal region of Kuma010. Since thesemutations lie within the propeptide domain, they are not present in themature, active enzyme. These two mutations fall within the N-terminalregion of the protein that sits in the enzyme's active site beforecleavage upon exposure to low pH. Since this region is in proximity ofthe catalytic residues, it is hypothesized that this is the region thatundergoes the initial cleavage event upon purification of the Kuma010enzyme during standard purification procedures. The partially cleavedenzyme N-terminus remains tightly associated with the mature enzymeuntil the enzyme is exposed to acid. While this initial self-processingduring protein purification does not negatively affect activity, it cancomplicate interpretation of SDS-PAGE analysis by individuals unfamiliarwith the Kuma010 enzyme. Thus, in order to simplify the SDS-PAGE profileof purified enzyme, the P171R and H172R mutations were incorporated toreduce the amount of initial N-terminal cleavage that occurs during theprotein purification process.

Since KumaXX1 and KumaXX2 variants lack a His tag, they are not purifiedby Ni affinity chromatography. Instead, these variants are purified byanion exchange chromatography. The following graph demonstrates activityof variants Kuma030, Kuma031, Kuma032, Kuma040, Kuma041, and Kuma042 onimmunogenic gliadin peptide DQ2.5-glia-α1a. In this case, all proteinswere purified by anion exchange chromatography (even Kuma030 and Kuma040which harbor an intact 6× Histidine tag), for the sake of comparison. Asshown in FIG. 7, P171R and H172R mutations do not appear to negativelyaffect activity as Kuma032 and Kuma042 demonstrate comparable activityas Kuma030 and Kuma040 (Kuma042 even shows a slight increase in activityagainst this substrate compared to Kuma040).

Kuma010 variant Kuma062 demonstrates a high amount of activity and itlacks a His tag. A comparison of Kuma062 with Kuma030 and Kuma040 isshown in FIGS. 8A-B (left, detection of full-length DQ2.5-glia-ω1;right, detection of breakdown product):

Enzyme Kinetics

Biochemical parameters were estimated for: Kuma010, Kuma030, Kuma040,and Kuma050. These were estimated using immunogenic gliadin epitopesDQ2.5-glia-α1a and DQ2.5-glia-ω1. Degradation assays were performed with100 nM enzyme at 37° C. in 100 mM NaOAc pH 4.0 buffer. The below tableshows the initial velocity of the degradation reaction as a function ofsubstrate concentration. k_(cat) and K_(M) were calculated from thisusing the Michaelis-Menten equation.

The biochemical parameters of all tested enzymes are shown in Table 7below.

TABLE 7 Enzyme [Enzyme] Substrate Vmax (M s⁻¹) Km (mM) Kcat (s⁻¹)kcat/Km (M⁻¹ s⁻¹) Kuma010 100 nM Glia_α1 3.70E−07  4 6 mM 3.7 819Kuma030 100 nM Glia_α1 1.74E−06  1.9 mM 17.4 9034 Kuma040 100 nM Glia_α11.49E−06 0.86 mM 16.3 19109 Kuma050 100 nM Glia_α1 1.42E−06  2.5 mM 14.25613 Kuma010 100 nM Glia_α1 1.22E−07 16.4 mM 1.2 74 Kuma030 100 nMGlia_α1 2.73E−06  8.4 mM 27.3 3268 Kuma040 100 nM Glia_α1 2.46E−06 15.3mM 24.6 1603 Kuma050 100 nM Glia_α1 2.78E−06  5.0 mM 27.8 56

The mutations made to Kuma030 and Kuma040 greatly increase activityagainst these peptides, which is promising for their use in detoxifyingpeptides that are linked to celiac disease. As predicted, the mutationsmade to Kuma050 increased activity on the DQ2.5-glia-α1a peptide, butnot on the DQ2.5-glia-ω1 peptide.

REFERENCES FOR EXAMPLE 2

(1) Gordon, S. R.; Stanley, E. J.; Wolf, S.; Toland, A.; Wu, S. J.;Hadidi, D.; Mills, J. H.; Baker, D.; Pultz, I. S.; Siegel, J. B. Journalof the American Chemical Society 2012.

(2) Wlodawer, A.; Li, M.; Gustchina, A.; Tsuruoka, N.; Ashida, M.;Minakata, H.; Oyama, H.; Oda, K.; Nishino, T.; Nakayama, T. J Biol Chem2004, 279, 21500.

(3) Leaver-Fay, A.; Tyka, M.; Lewis, S. M.; Lange, O. F.; Thompson, J.;Jacak, R.; Kaufman, K.; Renfrew, P. D.; Smith, C. A.; Sheffler, W.;Davis, I. W.; Cooper, S.; Treuille, A.; Mandell, D. J.; Richter, F.;Ban, Y. E.; Fleishman, S. J.; Corn, J. E.; Kim, D. E.; Lyskov, S.;Berrondo, M.; Mentzer, S.; Popovic, Z.; Havranek, J. J.; Karanicolas,J.; Das, R.; Meiler, J.; Kortemme, T.; Gray, J. J.; Kuhlman, B.; Baker,D.; Bradley, P. Methods Enzymol 2011, 487, 545.

(4) Wolf, C.; Siegel, J. B.; Tinberg, C.; Camarca, A.; Gianfrani, C.;Paski, S.; Guan, R.; Montelione, G.; Baker, D.; Pultz, I. S. Journal ofthe American Chemical Society 2015, 137, 13106.

(5) Shan, L.; Molberg, O.; Parrot, I.; Hausch, F.; Filiz, F.; Gray, G.M.; Sollid, L. M.; Khosla, C. Science 2002, 297, 2275.

(6) Shan, L. Journal of Proteome Research 2005.

(7) Sollid, L. M.; Qiao, S. W.; Anderson, R. P.; Gianfrani, C.; Koning,F. Immunogenetics 2012, 64, 455.

(8) Arentz-Hansen, H.; Korner, R.; Molberg, O.; Quarsten, H.; Vader, W.;Kooy, Y. M.; Lundin, K. E.; Koning, F.; Roepstorff, P.; Sollid, L. M.;McAdam, S. N. J Exp Med 2000, 191, 603.

We claim:
 1. A polypeptide comprising an amino acid sequence of SEQ IDNO:129.
 2. An isolated nucleic acid encoding the polypeptide of claim 1.3. A nucleic acid expression vector comprising the isolated nucleic acidof claim
 2. 4. A recombinant host cell comprising the nucleic acidexpression vector of claim
 3. 5. A pharmaceutical composition,comprising the polypeptide of claim 1, and a pharmaceutically acceptablecarrier.
 6. A method for treating celiac sprue, comprising administeringto an individual with celiac sprue an amount effective to treat theceliac sprue of the polypeptide of claim
 1. 7. A method for treatingceliac sprue, comprising administering to an individual with celiacsprue an amount effective of the pharmaceutical composition of claim 5to treat the celiac sprue.
 8. The method of claim 7, wherein thepolypeptide is administered orally.
 9. A polypeptide comprising an aminoacid sequence at least 99% identical to SEQ ID NO:59.
 10. An isolatednucleic acid encoding the polypeptide of claim
 9. 11. A nucleic acidexpression vector comprising the isolated nucleic acid of claim
 10. 12.A recombinant host cell comprising the nucleic acid expression vector ofclaim
 11. 13. A pharmaceutical composition, comprising the polypeptideof claim 9, and a pharmaceutically acceptable carrier.
 14. A method fortreating celiac sprue, comprising administering to an individual withceliac sprue an amount effective to treat the celiac sprue of thepolypeptide of claim
 9. 15. A method for treating celiac sprue,comprising administering to an individual with celiac sprue an amounteffective of the pharmaceutical composition of claim 13 to treat theceliac sprue.
 16. A polypeptide comprising an amino acid sequence atleast 99% identical to SEQ ID NO:59, wherein said amino acid sequencecomprises SEQ ID NO:129.
 17. An isolated nucleic acid encoding thepolypeptide of claim
 16. 18. A nucleic acid expression vector comprisingthe isolated nucleic acid of claim
 17. 19. A recombinant host cellcomprising the nucleic acid expression vector of claim
 18. 20. Apharmaceutical composition, comprising the polypeptide of claim 16, anda pharmaceutically acceptable carrier.
 21. A method for treating celiacsprue, comprising administering to an individual with celiac sprue anamount effective to treat the celiac sprue of the polypeptide of claim16.
 22. A method for treating celiac sprue, comprising administering toan individual with celiac sprue an amount effective of thepharmaceutical composition of claim 21 to treat the celiac sprue.
 23. Apolypeptide comprising an amino acid sequence at least 99% identical toSEQ ID NO:59, which includes at least one amino acid deletion from SEQID NO:59.
 24. An isolated nucleic acid encoding the polypeptide of claim23.
 25. A nucleic acid expression vector comprising the isolated nucleicacid of claim
 24. 26. A recombinant host cell comprising the nucleicacid expression vector of claim
 25. 27. A pharmaceutical composition,comprising the polypeptide of claim 23, and a pharmaceuticallyacceptable carrier.
 28. A method for treating celiac sprue, comprisingadministering to an individual with celiac sprue an amount effective totreat the celiac sprue of the polypeptide of claim
 23. 29. A method fortreating celiac sprue, comprising administering to an individual withceliac sprue an amount effective of the pharmaceutical composition ofclaim 27 to treat the celiac sprue.
 30. The method of claim 29, whereinthe polypeptide is administered orally.